Morphological identification of hookworm species in five regions of Cameroon

The soil-transmitted helminths (STH) are a group of parasitic nematode worms causing human infection through contact with parasite eggs or larvae that thrive in tropical and subtropical countries' warm and moist soil, but also in other climatic zones. Three main STH invasions, ascariasis, trichuriasis, and hookworms, are considered together because they are common for individuals. Hookworm invasions are currently considered one of the most underfunded neglected tropical diseases (NTDs). According to WHO's recent estimates, more than 1.5 billion people, or 24 % of the world's population, are infected with soil-transmitted helminth worldwide. Over 260 million preschool-age children, 654 million school-age children, 108 million adolescent girls and 138.8 million pregnant and lactating women living in areas where these parasites are intensively transmitted require treatment and preventive interventions (WHO, 2022). The burden of disease from soil-transmitted helminths is primarily attributable to their insidious and chronic impact on the health and quality of life of those infected rather than the mortality they cause. High-intensity infections caused the degradation of nutritional status and cognitive processes and were responsible for some 1.9 million disability-adjusted life years (DALYs) lost in 2019 (Levecke et al., 2014; Montresor et al., 2022). Many people are infected with hookworms without exhibiting symptoms of the disease. Heavy hookworm invasions in humans are associated with pathologic effects, including protein deficiency, heart failure, delayed puberty and mental dullness. Generally, hookworm disease is exhibited in cutaneous, pulmonary, and intestinal phases, most occurring in children and pregnant women (Schmidt et al., 2009; Vercruysse et al., 2011). Hookworms also cause and contribute to iron deficiency anaemia, which can negatively impact the health of children and women of childbearing age, foetuses, and newborn babies (Christian et al., 2004; Bethony et al., 2006).

Most diagnoses and research on the epidemiology of human hookworm invasion rely on using a conventional method to detect eggs in stool samples (Tchuem Tchuente et al., 2011, 2013). The benefits of this method are mainly technical simplicity and low cost. Although microscopy egg detection is limited and hampered because Necator americanus eggs are similar and morphologically indistinguishable from Ancylostoma spp., it is still the gold standard technique for rapidly diagnosing hookworms (Hawdon et al., 1996). Identifying the particular species prevalent in an endemic area is of utmost importance in epidemiological studies because of differences in the pathogenicity and egg-laying capacities of the two species. A female of A. duodenale lays about twenty thousand eggs daily, while N. americanus counterpart produces about ten thousand eggs per day (Chesbrough, 1992). Currently, mass treatments with anthelminthic drugs are performed without identifying the causative species of hookworm infections. Given that a clinical manifestation such as the severity of anaemia differs according to the hookworm species involved, a single A. duodenale ingests about 0.15 mL of human blood daily while one N. americanus ingest about 0.03 mL of blood within the same period (Beaver et al., 1984; Chesbrough, 1992). In the human intestine, A. duodenale lives for 1 – 3 years and N. americanus for 3 – 10 years with a maximum lifespan of 18 years. It has also been reported that A. duodenale is capable of entering latency within the host when the environment is unsuitable for its development and can be transmissible by oral, transplacental and lactogenic routes unlike N. americanus (Kumar & Pritchard, 1992; Brooker et al., 2004). In addition, diagnosis by precise identification and differentiation of species involved is essential in monitoring the efficacy of mass treatment and effective control of hookworm invasion (Akpan & Agida, 2013; Ngui et al., 2012). They can be easily differentiated by using copro-culture diagnostic tools as the stool culture followed by the morphological identification of infective third filariform (L3) larvae or by using molecular techniques such as PCR, PCRRFLP, and qPCR developed for molecular identification of STH species (Traub et al., 2004; Bethony et al., 2006; Areekul et al., 2010; George et al., 2015; Ng-Nguyen et al., 2015).

In Cameroon, most studies did not attempt to differentiate and relied on past epidemiological data, which indicates the predominance of N. americanus infections (Brooker et al., 2000; Tchuem Tchuente et al., 2003). Recent studies based on the identification of L3 larvae recovered from the stool of school children after culture showed the occurrence of A. duodenale and mixed infections with N. americanus in one of the ten regions of Cameroon (Kamwa, 2012). Similar observations were made in neighbouring Nigeria, where N. americanus was previously described as the only endemic species, but a study on the morphological identification of L3 larvae allows to describe A. duodenale (Adenusi, 1997; Adenusi & Ogunyomi, 2003). Therefore, this study aimed to determine the distribution of the two hookworm species in five regions of Cameroon.

The protocol of this study was approved, and ethical clearances were obtained from the National Ethics Committee of Cameroon (N° 082/CNE/DNM/09, N° 147/CNE/DNM11). Before data collection, the approvals of administrative authorities were obtained. The study's objectives were explained to the school children, parents, and guardians from whom informed consent was obtained and signed.

The identification criteria for hookworm L3 larvae cultured by the Harada-Mori method were based on the distinction of nematode larvae's morphological characters as WHO described (1981). Among the identification keys of nematode larvae's morphological characters, only three were clearly visible on the identical larvae, allowing us to identify the species. These are the mouth, the oesophagus-intestine junction, and the tail morphology. Figures 2 and 3 display some morphological details of L3 filariform larvae observed on hookworm-positive stool samples after culture in our study, showing the differences between N. americanus and A. duodenale. In N. americanus, the sheath around the rounded head, the gap between the intestine and the oesophagus, and the sharply pointed tail with striations are pretty visible. A. duodenale is distinguishable from N. americanus with the blunt head and tail and the absence of a gap between the intestine and the oesophagus. From the 275 schools investigated, 157 positive stool samples were collected in 47 schools distributed unequally in the five regions. They were cultured by the Harada-Mori method with an overall sensibility rate of 57 %. The sensibility rate increased as we moved away from hot regions; the highest sensitivity rate was observed in the Littoral (76.47 %) and the South-West regions (71.11 %). Out of 8061 L3 larvae characterized, 230 (2.85 %) were identified as A. duodenale and 7831 (97.15 %) as N. americanus (Table 2). A. duodenale has only been found in the Littoral region, only in the Mouanko health district, where past morphological identification has indicated its occurrence. However, during our study, from the 157 stool samples cultured, we registered 18 cases of N. americanus and A. duodenale mixed infections (11.46 %) (Fig. 4).

Fig. 2.

Head of a hookworm L3 larvae recovered from a hookworm-positive faecal sample

Fig. 3.

Tail of a hookworm L3 larvae recovered from a hookworm-positive faecal sample.

Fig. 4.

Hookworm entire L3 larvae recovered from hookworm-positive faecal samples.

From this study, the results of the morphological identification of hookworm species in five regions of Cameroon using the Harada-Mori stool culture approach show that the sensitivity to the Harada-Mori test differs in the five regions and increases as we move away from the warmer areas. This could be justified by the fact that hookworm species thrive in the optimal temperatures of the larval stages located around 22 – 27°C for A. duodenale and 28 – 30 °C for N. americanus (Crompton & Whitehead, 1993; Hossain & Bhuiyan, 2016). Following the criteria for species identification described by WHO (1981) and Yoshida (1966), morphological identification of the L3 larvae obtained after stool culture shows the presence of two distinct morphological forms, one belonging to N. americanus and the other to A. duodenale. The simultaneous presence of both species was observed only in the Littoral region. This result corroborates the findings of Kamwa (2012) in the same locality of Mouanko, where he reported the presence of N. americanus (50 %), A. duodenale (47.06 %) and cases of mixed infection by both species (14.74 %). In our study, we also registered 11.46 % of mixed infections. Both A. duodenale and N. americanus infections have been reported in humans in northern Ghana and neighbouring Nigeria after stool culture and identification of the L3 larvae stage (Adenusi & Ogunyomi, 2003; Kwabena, 2009). Our findings also imply that when hookworm infection is diagnosed in the Far-North, North, Adamawa and South-West regions, it is most likely N. americanus infection. However, because of some factors, such as the low number of samples collected per site, the lack of suitable equipment for on-site identification, and the temperature variation observed during the transfer of the samples from the site to the laboratory during incubation, the data available cannot be considered sufficient to conclude on the absence of A. duodenale infection among hookworm-positive school children reporting at these four regions. In addition to these factors, the low yield of the Harada-Mori method compared to other soil-transmitted helminth culture methods, such as the Baermann method and the Agar plate culture technique, could be considered. Indeed, a comparative study of these methods carried out by Reiss et al. (2007) showed that the yield of viable larvae from the Agar plate and Baermann methods was comparable (50 % and 47 %, respectively) but greater than that of the Harada-Mori method (2.1 %). Recent work carried out on the sensitivity of the Harada-Mori method compared to the previous method also shows that the sensitivity rate is significantly lower than the sensitivity rates of the Agar plate and Baermann method (Blatt & Cantos, 2003; Nongmaithem et al., 2019).

According to the molecular study conducted by George et al. (2016), it is not fully understood why A. duodenale was not found in the South-West region, especially in Ekondo-Titi, because it was reported to be relatively common in Ekondo-Titi, with a 35 % over 7.5 % prevalence for N. americanus. It is possible that our samples were not from the same schools investigated by George et al. (2016) or that our data were insufficient to conclude the absence of A. duodenale. An extended molecular study in this locality and the localities where only N. americanus has been identified will be necessary to conclude the distribution of the different species in Cameroon firmly. Moreover, A. duodenale is reported to differ in susceptibility to the same anthelminthic and dosage regimen (Rim et al., 1971; Reynoldson et al., 1997). Identifying the type of hookworm species being transmitted in a particular community is essential because it influences the burden of iron deficiency anaemia in the community. So, in a community with Ancylostoma species, radical treatment and efficient management of anaemia will be required. The difference in susceptibility of the two hookworm species for the same anthelmintic drug mentioned above refers to pyrantel pamoate and bephenium hydroxynaphthoate, two drugs currently out of use in mass deworming campaigns. However, no differential efficacy study has ever been reported on Mebendazole and Albendazole, two drugs currently used in the control of STH and whose efficacy for hookworms has always been lower than that of Ascaris (Vercruysse et al., 2010; Montresor et al., 2022). A subsequent study on this differential efficacy will be conducted. It is also important to point out that the areas where A. duodenale was identified by morphological identification (Mouanko in the Littoral region), and molecular tools (Ekondo-Titi in the South-West region) are near Nigeria's border. The similar geographic conditions in these regions could explain this. There is geographic variance in the distribution of the two human hookworm species, which is a multi-factorial phenomenon, given that human and parasite behavior, ethnicity, climate, temperature, and environmental factors are involved (Hoagland & Schad, 1978). In the northern regions of Cameroon (Far-North, North and Adamawa), the results of the mapping activities show a low prevalence and intensity of the soil-transmitted helminths, but this low endemicity alone could not explain the absence of A. duodenale in these regions. A molecular study should be considered to conclude the distribution of hookworms in the northern regions and to establish the role of animal parasites in the endemicity of STH in Cameroon.

Hookworm infection is endemic in all the five regions investigated in our study. The Harada-Mori culture method allowed us to morphologically distinguish two species of hookworms with a predominance for N. americanus. A. duodenale remains endemic in the health district of Mouanko in the Littoral region. A molecular study is obvious to reach more conclusions on the distribution of these species in Cameroon.